Generation of biologically active angiostatin kringle 1-3 by activated human neutrophils

The contribution of polymorphonuclear neutrophils (PMN) to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. In this study, we demonstrate that upon stimulation with proinflammatory stimuli, human PMN release enzymatic activities that, in vitro, generate bioactive angiostatin fragments from purified plasminogen. We also provide evidence that these angiostatin-like fragments, comprising kringle domain 1 to kringle domain 3 (kringle 1-3) of plasminogen, are generated as a byproduct of the selective proteolytic activity of neutrophil-secreted elastase. Remarkably, affinity-purified angiostatin kringle 1-3 fragments generated by neutrophils inhibited basic fibroblast growth factor plus vascular endothelial growth factor-induced endothelial cell proliferation in vitro, and both vascular endothelial growth factor-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo. These results represent the first demonstration that biologically active angiostatin-like fragments can be generated by inflammatory human neutrophils. Because angiostatin is a potent inhibitor of angiogenesis, tumor growth, and metastasis, the data suggest that activated PMN not only act as potent effectors of inflammation, but might also play a critical role in the inhibition of angiogenesis in inflammatory diseases and tumors, by generation of a potent anti-angiogenic molecule.     

The role of angiostatin in the spontaneous bone and prostate cancers of pet dogs

Angiostatin is a potent inhibitor of angiogenesis generated in cancer-bearing hosts by tumor-derived proteases. Because the naturally occurring bone and prostate cancers of pet dogs provide unique model systems to study factors that regulate cancer progression and tumor dormancy, we investigated the capacity of these tumors to generate angiostatin. We determined that angiostatin fragments are present in urine of dogs with bone cancer. The identity of these fragments was confirmed by comparison of the experimentally determined protein sequence to that of a clone of canine angiostatin. Importantly, these fragments were absent in urine collected from the same dogs after complete surgical removal of the primary tumor. We also demonstrate that canine prostate cancer cells are capable of processing plasminogen to angiostatin in vitro. These findings provide rationale for using spontaneous canine tumor models to isolate endogenous angiogenesis inhibitors and to investigate their therapeutic use against cancer.     

Angiostatin's molecular mechanism: aspects of specificity and regulation elucidated

Tumor growth requires the development of new vessels that sprout from pre-existing normal vessels in a process known as "angiogenesis" [Folkman (1971) N Engl J Med 285:1182-1186]. These new vessels arise from local capillaries, arteries, and veins in response to the release of soluble growth factors from the tumor mass, enabling these tumors to grow beyond the diffusion-limited size of approximately 2 mm diameter. Angiostatin, a naturally occurring inhibitor of angiogenesis, was discovered based on its ability to block tumor growth in vivo by inhibiting the formation of new tumor blood vessels [O'Reilly et al. (1994a) Cold Spring Harb Symp Quant Biol 59:471-482]. Angiostatin is a proteolytically derived internal fragment of plasminogen and may contain various members of the five plasminogen "kringle" domains, depending on the exact sites of proteolysis. Different forms of angiostatin have measurably different activities, suggesting that much remains to be elucidated about angiostatin biology. A number of groups have sought to identify the native cell surface binding site(s) for angiostatin, resulting in at least five different binding sites proposed for angiostatin on the surface of endothelial cells (EC). This review will consider the data supporting all of the various reported angiostatin binding sites and will focus particular attention on the angiostatin binding protein identified by our group: F(1)F(O) ATP synthase. There have been several developments in the quest to elucidate the mechanism of action of angiostatin and the regulation of its receptor. The purpose of this review is to describe the highlights of research on the mechanism of action of angiostatin, its' interaction with ATP synthase on the EC surface, modulators of its activity, and issues that should be explored in future research related to angiostatin and other anti-angiogenic agents.     

Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity

Bone is a very common metastatic site for breast cancer. In bone metastasis, there is a vicious circle wherein bone-residing metastatic cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone promote tumor growth. The contribution of tumor angiogenesis in the growth of bone metastases is, however, unknown. By using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells that quite closely mimics the conditions likely to occur in naturally arising metastatic human breast cancers, we demonstrate here that when MDA-MB-231/B02 cells were engineered to produce at the bone metastatic site an angiogenesis inhibitor, angiostatin, there was a marked inhibition in the extent of skeletal lesions. Inhibition of skeletal lesions came with a pronounced reduction in tumor burden in bone. However, although angiostatin produced by MDA-MB-231/B02 cells was effective at inhibiting in vitro endothelial cell proliferation and in vivo angiogenesis in a Matrigel implant model, we have shown that it inhibited cancer-induced bone destruction through a direct inhibition of osteoclast activity and generation. Overall, these results indicate that, besides its well known anti-angiogenic activity, angiostatin must also be considered as a very effective inhibitor of bone resorption, broadening its potential clinical use in cancer therapy.     

Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation

Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type-specific manner. We have used a construct encoding the kringle domains 1--4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.     

Angiostatin is negatively associated with coronary collateral growth in patients with coronary artery disease

Angiostatin, an inhibitor of tumor angiogenesis, is produced by the actions of matrix metalloproteinases (MMP) on plasminogen. Recently, we reported that angiostatin levels are increased in a model of inadequate coronary collateral growth and angiogenesis in response to ischemia, despite high levels of vascular endothelial growth factor (VEGF). We hypothesized that angiostatin levels are negatively associated with collateral formation in patients. Coronary angiograms from 37 patients undergoing coronary bypass surgery were evaluated for the absence of angiographically visible collaterals (Rentrop scores of 0) or the presence of Rentrop classification grade 3 (well developed) collaterals. Pericardial fluid was obtained from each patient during the bypass procedure, and the sample was analyzed for angiostatin, plasminogen, and VEGF (Western analysis) and for combined activities of MMP-2 and MMP-9 (zymographic analysis). In patients with no collaterals, angiostatin level was greater compared with that in patients with well-developed collaterals (3.1 +/- 0.2 vs. 2.3 +/- 0.1 optical density units, P < 0.05). Neither MMP activities nor VEGF levels were different between the two groups of patients. The higher levels of angiostatin in patients with no visible collaterals were reflective of a higher concentration of plasmin/plasminogen (6.2 +/- 0.7 vs. 4.2 +/- 0.5 optical density units, P < 0.05) compared with those in patients with well-developed collateral vessels. Our results support the concept that the growth inhibitor angiostatin may have a negative impact on coronary collateral growth in patients. Perhaps therapies attempting to provoke coronary collateral growth should incorporate approaches to limit or neutralize the effects of growth inhibitors.     

Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells

Angiostatin, a potent endogenous inhibitor of angiogenesis, is generated by cancer-mediated proteolysis of plasminogen. The culture medium of human prostate carcinoma cells, when incubated with plasminogen at a variety of pH values, generated angiostatic peptides and miniplasminogen. The enzyme(s) responsible for this reaction was purified and identified as procathepsin D. The purified procathepsin D, as well as cathepsin D, generated two angiostatic peptides having the same NH(2)-terminal amino acid sequences and comprising kringles 1-4 of plasminogen in the pH range of 3.0-6.8, most strongly at pH 4.0 in vitro. This reaction required the concomitant conversion of procathepsin D to catalytically active pseudocathepsin D. The conversion of pseudocathepsin D to the mature cathepsin D was not observed by the prolonged incubation. The affinity-purified angiostatic peptides inhibited angiogenesis both in vitro and in vivo. Importantly, procathepsin D secreted by human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that by human prostate carcinoma cells. Since deglycosylated procathepsin D from both prostate and breast carcinoma cells exhibited a similar low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in carbohydrate structures of procathepsin D molecules between the two cell types. The seminal vesicle fluid from patients with prostate carcinoma contained the mature cathepsin D and procathepsin D, but not pseudocathepsin D, suggesting that pseudocathepsin D is not a normal intermediate of procathepsin D processing in vivo. The present study provides evidence for the first time that cathepsin D secreted by human prostate carcinoma cells is responsible for angiostatin generation, thereby causing the prevention of tumor growth and angiogenesis-dependent growth of metastases.     

Limited Proteolysis of Angiogenin by Elastase Is Regulated by Plasminogen

Human neutrophil elastase cleaves angiogenin at the Ile-29/Met-30 peptide bond to produce two major disulfide-linked fragments with apparent molecular weights of 10,000 and 4000, respectively. Elastase-cleaved angiogenin has slightly increased ribonucleolytic activity, but has lost its ability to undergo nuclear translocation in endothelial cells, a process essential for angiogenic activity. Cleavage appears to alter the cell-binding properties of angiogenin, despite the fact that it occurs some distance from the putative receptor-binding site, since the elastase-cleaved protein fails to compete with its native counterpart for nuclear translocation in endothelial cells. Plasminogen specifically accelerates elastase proteolysis of angiogenin. It does not enhance elastase activity toward ribonuclease A or the synthetic peptide substrate MeOSuc-Ala-Ala-Pro-Val-pNA. Plasminogen-accelerated inactivation of angiogenin by elastase might be a significant event in the process of angiogenin-induced angiogenesis since (i) angiogenin and plasminogen circulate in plasma at high concentrations, (ii) angiogenin, especially when bound to actin, activates tissue plasminogen activator to generate plasmin from plasminogen, and (iii) elastase cleaves plasminogen to produce angiostatin, a potent inhibitor of angiogenesis and metastasis. Interrelationships among angiogenin, plasminogen, plasminogen activators, elastase, and angiostatin may provide a sensitive regulatory system to balance angiogenesis and antiangiogenesis.     

Angiostatin directly inhibits human prostate tumor cell invasion by blocking plasminogen binding to its cellular receptor, CD26

Previous studies demonstrate that one of the six plasminogen type 2 glycoforms, plasminogen 2epsilon, enhances invasiveness of the 1-LN human prostate tumor cell line in an in vitro model. Binding of plasminogen 2epsilon to CD26 on the cell surface induces a Ca(2+) signaling cascade which stimulates the expression of matrix metalloproteinase-9, required by these cells to invade Matrigel. We now report that angiostatin, a fragment derived from plasminogen which prevents endothelial cell proliferation, is also a potent, direct inhibitor of 1-LN tumor cell invasiveness. We studied the effect of individual plasminogen 2 glycoform-derived angiostatins and found that only angiostatin 2epsilon binds to CD26 on the surface of 1-LN cells at a site also recognized by plasminogen 2epsilon. As a result, the plasminogen 2epsilon-induced Ca(2+) signaling cascade is inhibited, the expression of matrix metalloproteinase-9 is suppressed, and invasion of Matrigel by 1-LN cells is blocked. Angiostatin 2epsilon is also the only angiostatin glycoform which is able to inhibit in vitro endothelial cell proliferation and tubule formation. These studies suggest that, in addition to its ability to inhibit tumor vascularization, angiostatin 2epsilon may also directly block tumor metastasis.     

Specific interaction of angiostatin with integrin alpha(v)beta(3) in endothelial cells

Angiostatin, the N-terminal four kringles (K1-4) of plasminogen, blocks tumor-mediated angiogenesis and has great therapeutic potential. However, angiostatin's mechanism of anti-angiogenic action is unclear. We found that bovine arterial endothelial (BAE) cells adhere to angiostatin in an integrin-dependent manner and that integrins alpha(v)beta(3), alpha(9)beta(1), and to a lesser extent alpha(4)beta(1), specifically bind to angiostatin. alpha(v)beta(3) is a predominant receptor for angiostatin on BAE cells, since a function-blocking antibody to alpha(v)beta(3) effectively blocks adhesion of BAE cells to angiostatin, but an antibody to alpha(9)beta(1) does not. epsilon-Aminocaproic acid, a Lys analogue, effectively blocks angiostatin binding to BAE cells, indicating that an unoccupied Lys-binding site of the kringles may be required for integrin binding. It is known that other plasminogen fragments containing three or five kringles (K1-3 or K1-5) have an anti-angiogenic effect, but plasminogen itself does not. We found that K1-3 and K1-5 bind to alpha(v)beta(3), but plasminogen does not. These results suggest that the anti-angiogenic action of angiostatin may be mediated via interaction with alpha(v)beta(3). Angiostatin binding to alpha(v)beta(3) does not strongly induce stress-fiber formation, suggesting that angiostatin may prevent angiogenesis by perturbing the alpha(v)beta(3)-mediated signal transduction that may be necessary for angiogenesis.     

Inhibition of endothelial cell proliferation by the recombinant kringle domain of tissue-type plasminogen activator

Tissue-type plasminogen activator (tPA) is a multidomain serine protease that converts the zymogen plasminogen to plasmin. tPA contains two kringle domains which display considerable sequence identity with those of angiostatin, an angiogenesis inhibitor. TK1-2, a recombinant kringle domain composed of t-PA kringles 1 and 2 (Ala(90)-Thr(263)), was produced by both bacterial and yeast expression systems. In vitro, TK1-2 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor. It did not inhibit proliferation of non-endothelial cells. TK1-2 also inhibited in vivo angiogenesis in the chick embryo chorioallantoic membrane model. These results suggest that the recombinant kringle domain of t-PA is a selective inhibitor of endothelial cell growth and identifies this molecule as a novel anti-angiogenic agent.     

Interaction of plasminogen-related protein B with endothelial and smooth muscle cells in vitro

Plasminogen-related protein B (PRP-B) closely resembles the N-terminal plasminogen activation peptide, which is released from plasminogen during conversion to plasmin. We have previously demonstrated that the steady-state level of mRNA encoding PRP-B is increased within tumor tissues, and that recombinant PRP-B antagonizes neoplastic growth when administered systemically to mice harboring tumors, but no insights into the cell targets of PRP-B have been presented. Employing serum-free medium optimized for culturing human endothelial or smooth muscle cells, we show that recombinant PRP-B inhibits basic fibroblast growth factor-dependent cell migration for both cell types, as well as tube formation of endothelial cells. Comparison with the angiogenesis inhibitors angiostatin and endostatin revealed similar results. Recombinant PRP-B is effective in promoting cell attachment of endothelial and smooth muscle cells, and antibody interference experiments reveal that the interaction of recombinant PRP-B with endothelial cells is mediated at least in part by alpha(v)-containing integrins. Inhibition of angiogenesis in vivo by PRP-B was demonstrated in the chicken chorioallantoic membrane assay. PRP-B and other antiangiogenic molecules may elicit metabolic perturbations in endothelial cells as well as perivascular mesenchymal cells such as smooth muscle cells and pericytes.     

Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis

Angiogenesis is a process of new blood vessel development from pre-existing vasculature and it plays an essential role in tumor growth and metastases. Here, we investigate the expression of muscarinic acetylcholine receptors (mAchR) and their participation in tumor cell proliferation and angiogenesis ability. Saturation binding assays with the tritiated muscarinic antagonist quinuclidinyl benzilate indicate that LMM3 cells derived from a murine mammary adenocarcinoma express a single class of functional mAchR. Competition binding assays with selective muscarinic antagonists indicate a predominance of M3 receptor subtype. The muscarinic agonist carbachol (CARB) stimulates LMM3 cell proliferation in a concentration dependent manner. The maximal effect induced by 10(-9)M CARB was totally blunted by atropine and by the selective M3 and M1 antagonists, para-fluoro hexahydro sila-difenidol (pf-HHSiD) and pirenzepine, respectively. In addition, pf-HHSiD completely blocked in vivo CARB-induced neovascular formation and vascular endothelial growth factor-A in LMM3 tumor cells. We can conclude that mAchR expressed in LMM3 mammary tumor cells positively regulate proliferation and angiogenesis required for tumor progression.     

A mathematical model for the roles of pericytes and macrophages in the initiation of angiogenesis. I. The role of protease inhibitors in preventing angiogenesis

In this paper, a simple mathematical model developed in H.A. Levine, B.D. Sleeman, M. Nilsen-Hamilton [J. Math. Biol., in press] to describe the initiation of capillary formation in tumor angiogenesis is extended to include the roles of pericytes and macrophages in regulating angiogenesis. The model also allows for the presence of anti-angiogenic (angiostatic) factors. The model is based on the observation that angiostatin can prevent the degradation of fibronectin in the basal lamina by inhibiting the catalytic action of active proteolytic enzyme. That is, it is proposed that the inhibitor 'deactivates' the protease but that it does not reduce the over all concentration of the protease. It consequently explores the possibility of preventing neovascular capillaries from migrating through the extra-cellular matrix toward the tumor by inhibiting protease action. The model is based on the theory of reinforced random walks coupled with Michaelis-Menten mechanisms which view endothelial cell receptors as the catalysts for transforming both tumor and macrophage derived angiogenic factors into proteolytic enzyme which in turn degrade the basal lamina. A simple catalytic reaction is proposed for the degradation of the basal lamina by the active proteases. A mechanism, in which the angiostatin acts as a protease inhibitor is discussed which has been substantiated experimentally. A second mechanism for the production of protease inhibitor from angiostatin by endothelial cells is proposed to be of Michaelis-Menten type. Mathematically, this mechanism includes the former as a subcase.     

Plasmin-induced migration of endothelial cells. A potential target for the anti-angiogenic action of angiostatin

Angiostatin, a plasminogen fragment containing 3-4 N-terminal kringle domains, is a potent inhibitor of tumor-induced angiogenesis, but its mechanism of action is unclear. Angiostatin is a ligand for integrin alphavbeta(3) but does not induce stress fiber formation upon integrin binding, suggesting that angiostatin is a potential integrin antagonist. Plasmin, the parent molecule of angiostatin and a major extracellular protease, induces platelet aggregation, migration of peripheral blood monocytes, and release of arachidonate and leukotriene from several cell types. In the current study, we found that plasmin specifically bound to alphavbeta(3) through the kringle domains and induced migration of endothelial cells. In contrast, angiostatin did not induce cell migration. Notably, angiostatin, anti-alphavbeta(3) antibodies, RGD-peptide, and a serine protease inhibitor effectively blocked plasmin-induced cell migration. These results suggest that plasmin-induced migration of endothelial cells requires alphavbeta(3) and the catalytic activity of plasmin and that this process is a potential target for the inhibitory activity of angiostatin.     

Annexin A2-S100A10 heterotetramer, a novel substrate of thioredoxin

The binding of plasminogen activators and plasminogen to the cell surface results in the rapid generation of the serine protease plasmin. Plasmin is further degraded by an autoproteolytic reaction, resulting in the release of an angiostatin, A61 (Lys78-Lys468). Previously, we demonstrated that the annexin A2-S100A10 heterotetramer (AIIt) stimulates the release of A61 from plasmin by promoting the autoproteolytic cleavage of the Lys468-Gly469 bond and reduction of the plasmin Cys462-Cys541 disulfide (Kwon, M., Caplan, J. F., Filipenko, N. R., Choi, K. S., Fitzpatrick, S. L., Zhang, L., and Waisman, D. M. (2002) J. Biol. Chem. 277, 10903-10911). Mechanistically, it was unclear if AIIt promoted a conformational change in plasmin, resulting in contortion of the plasmin disulfide, or directly reduced the plasmin disulfide. In the present study, we show that AIIt thiols are oxidized during the reduction of plasmin disulfides, establishing that AIIt directly participates in the reduction reaction. Incubation of HT1080 cells with plasminogen resulted in the rapid loss of thiol-specific labeling of AIIt by 3-(N-maleimidopropionyl)biocytin. The plasminogen-dependent oxidation of AIIt could be attenuated by thioredoxin. Thioredoxin reductase catalyzed the transfer of electrons from NADPH to the oxidized thioredoxin, thus completing the flow of electrons from NADPH to AIIt. Therefore, we identify AIIt as a substrate of the thioredoxin system and propose a new model for the role of AIIt in the redox-dependent processing of plasminogen and generation of an angiostatin at the cell surface.     

Temporal and Pharmacological Characterization of Angiostatin Release and Generation by Human Platelets: Implications for Endothelial Cell Migration

Platelets play an important role in thrombosis and in neo-vascularisation as they release and produce factors that both promote and suppress angiogenesis. Amongst these factors is the angiogenesis inhibitor angiostatin, which is released during thrombus formation. The impact of anti-thrombotic agents and the kinetics of platelet angiostatin release are unknown. Hence, our objectives were to characterize platelet angiostatin release temporally and pharmacologically and to determine how angiostatin release influences endothelial cell migration, an early stage of angiogenesis. We hypothesized anti-platelet agents would suppress angiostatin release but not generation by platelets. Human platelets were aggregated and temporal angiostatin release was compared to vascular endothelial growth factor (VEGF). Immuno-gold electron microscopy and immunofluorescence microscopy identified α-granules as storage organelles of platelet angiostatin. Acetylsalicylic acid, MRS2395, GPIIb/IIIa blocking peptide, and aprotinin were used to characterize platelet angiostatin release and generation. An endothelial cell migration assay was performed under hypoxic conditions to determine the effects of pharmacological platelet and angiostatin inhibition. Compared to VEGF, angiostatin generation and release from α-granules occurred later temporally during platelet aggregation. Consequently, collagen-activated platelet releasates stimulated endothelial cell migration more potently than maximally-aggregated platelets. Platelet inhibitors prostacyclin, S-nitroso-glutathione, acetylsalicylic acid, and GPIIb/IIIa blocking peptide, but not a P2Y12 inhibitor, suppressed angiostatin release but not generation. Suppression of angiostatin generation in the presence of acetylsalicylic acid enhanced platelet-stimulated endothelial migration. Hence, the temporal and pharmacological modulation of platelet angiostatin release may have significant consequences for neo-vascularization following thrombus formation.     

Angiostatin antagonizes the action of VEGF-A in human endothelial cells via two distinct pathways

Angiostatin consisting of the first four-kringle domains of the plasminogen potently inhibits angiogenesis in vitro and in vivo. However, the molecular mechanism of action whereby angiostatin mediates its inhibitory effect on proliferating endothelial cells remains elusive. We therefore used the proliferating cultured human umbilical vein endothelial cells (HUVECs) promoted by vascular endothelial growth factor A to identify the endogenous signaling elements that mediate the antiangiogenic effect of angiostatin. Treatment of HUVEC with angiostatin at a concentration known to inhibit cell proliferation and induce apoptosis resulted in induction of p53-, Bax-, and tBid-mediated release of cytochrome c into the cytosol. In addition, angiostatin also activated the Fas-mediated apoptotic pathway in part via up-regulation of FasL mRNA, down-regulation of c-Flip, and activation of caspase 3. These results suggest that the anti-angiogenic action of angiostatin is likely mediated by two distinct signaling pathways, one intrinsic mediated by p53 while the other extrinsic involved in FasL engagement and mitochondria dysfunction.     

Mechanism of the inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5, a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis, on kinetic parameters (k _Pg and K _Pg) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect on the k _Pg value, but increases the value of K _Pg plasminogen activation by urokinase. A decrease in the k _Pg value and an increase in the K _Pg value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is a competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA with a mixed type. Such an influence of angiostatin on the kinetic constants of the plasminogen activation by urokinase suggests that angiostatin dose-dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In the case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with the formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex, which leads to a decrease in k _Pg and an increase in K _Pg of the plasminogen activation. Inhibition constants by angiostatin (K _i) of plasminogen-activator activities of uPA and tPA determined by the Dixon method were found to be 0.59 ± 0.04 and 0.12 ± 0.05 μM, respectively.     

Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides.